Simvastatin inhibits plaque rupture and subsequent thrombus formation in atherosclerotic rabbits with hyperlipidemia
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《中华医药杂志》英文版
【Abstract】 Objective To observe the effects of simvastatin on plaque stability and its possible mechnism in atherosclerotic rabbits. Methods Male New Zealand rabbits were fed with a high fat diet for 8 weeks and a regular diet followed for 8 weeks. At 2 weeks after high fat diet, abdominal aortic endothelium was damaged by using 4F Forgarty catheter, at the 5th week after regular diet, the rabbits were administered with simvastatin 1mg/(kg·d) for 4 weeks, then the rabbit atherosclerotic plaque rupture and thrombosis were triggered by injection of Russells viper venom (RVV, 0.12mg/kg,ip.) and histamine (0.016mg/kg,iv.). Area of thrombosis on atherosclerotic aorta, morphologic character of plaque rupture, levels of blood lipid, contents of lipid, 6-keto-prostaglandin F1α(6-keto-PGF1α), thromboxane B2 (TXB2) and matrix metalloproteinase (MMP)-2 mRNA expression in aorta were determined by image analysis, light and electron microscopes, chromatometry, radioimmunoassay and hybridization in situ, respectively. Results The surface area covered by thrombus was (0.9±1.1)mm2 in control group,(78±53 )mm2 in model group, and (17±12)mm2 in simvastatin-treated group. Arterial plaque in model group showed obvious ulcers occurred and inflammatory cells infiltrated in shoulder area of plaque. Fibre cap on plaque in simvastatin-treated group was thicker and more integrant than that in model group, and inflammatory cell infiltration was also decreased. Compared with model group, serum total cholesterol (TC), triglyceride (TG) and low density lipoprotein-cholesterol (LDL-C) in simvastatin-treated group were decreased by 24.8 %, 39.9 % and 21.1 %, respectively, contents of cholesterol in abdominal aorta and TXB2 in thoracic aorta were decreased by 45.8 % and 24.2 %, respectively, while level of 6-keto-PGF1α and ratio of 6-keto-PGF1α/ TXB2 in aorta were significantly increased. MMP-2 mRNA in abdominal aorta expressed less in simvastatin-treated group than in model group.Conclusions Simvastatin could be an increase plaque stability and inhibit thrombosis, its action mechanisms might be associated with inhibition of inflammatory cell and lipid infiltration, MMP-2 mRNA expression, and thromboxane A2 (TXA2) formation, and increment of prostacyclin (PGI2) production and PGI2/TXA2 ratio in atherosclerotic plaque.
【Key words】 simvastatin; plaque stability; thrombosis; rabbits
INTRODUCTION
Cardio-cerebral vascular disease is the leading cause of mortality in many countries and carries an important socioeconomic burden. Atherosclerosis is the main pathologic basis of cardio-cerebral vascular disease. With the development of atherosclerosis, plaque rupture and thrombosis will occur, that is critical to the onset of stroke, acute coronary ischemic syndromes[1-3] and so on. The vulnerable plaque is generally composed of an atrophic fibrous cap, a lipid-rich necrotic core, and accumulation of inflammatory cells[4-7]. Erosion of the fibrous cap, usually correlating to matrix metalloproteinases (MMPs) [8,9] such as MMP-2, often leads to rupture of the plaque, with subsequent occlusive thrombus formation, which relates to the imbalance between PGI2 and TXA2[10-13].PGI2 generated by vascular endotheliocytes, is a potent platelet inhibitor and vasodilator, and TXA2, released from activated platelets, is a potent vasoconstrictor and platelet-aggregating agent. Recent research has identified that the stability of plaque is more significant than its absolute size[14-16], so it is crucial to increase plaque stability to prevent acute cardiovascular and cerebrovascular diseases.
Statins such as simvastatin, a kind of lipidemic regulators, are widely used for the treatment of hypercholesterolaemia. In recent years, however, there has been an increasing body of evidence that their effects on lipid profile cannot fully account for their cardiovascular protective actions[17-20]: their beneficial effects are too rapid to be easily explained by their relatively slow effects on atherogenesis and too large to be accounted for by their relatively small effects on plaque regression.Therefore,it is now thought that their beneficial effects on cardio-cerebral vascular system might result from improving vascular endothelial function, inhibiting platelet aggregation, and reducing inflammatory cell accumulation[21-24]. These findings of clinical observation and cell culture prompted us to investigate whether statins increased plaque stability in animal model of atherosclerotic plaque rupture.
The present studies were designed to address the following three questions. First, we wished to determine whether simvastatin had any effect on reducing thrombotic area in rabbit atherosclerotic plaque rupture model. Secondly, we wished to evaluate changes in the morphological features related to plaque instability after treatment of atherosclerotic rabbits with simvastatin. Finally, we wished to determine whether simvastatin had any effect on TXA2, PGI2 and MMP-2 in aortic vessel tissue. The data demonstrated that simvastatin not only reduced lipid levels, but also inhibited plaque repture and subsequent thrombosis, its mechanisms might include decreasing inflammatory cell and lipid infiltration, MMP-2 mRNA expression and TXA2 formation, increasing PGI2 content and PGI2/TXA2 ratio in the artery covered with atherosclerotic plaque.
MATERIALS AND METHODS
Animals:Male New Zealand white rabbits, weighing (2.0±0.2)kg were provided by Experimental Animal Center of Soochow University (Certificate No: YCYK 2002-0008).
Drugs and reagents:Simvastatin was provided by Hangzhou Merck Sharp & Dohme Pharmaceutical Co. Ltd, China. Russells viper venom (RVV) and histamine were products of Sigma Chemical Co，USA. Cholesterol was a product of Nanjing Xinbai Pharmaceutical Co., China. TC, TG, high density lipoprotein-cholesterol (HDL-C) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. MMP-2 mRNA in situ hybridization detection kit was supplied by Wuhan Boshide Bioengineering Co. Ltd, China. 6-keto-PGF1α and TXB2 radioimmunity assay kits were supplied by Radioimmunity Institute of Soochow University.
Animal model preparation:Experimental rabbits were fed with a high fat diet (cholesterol 0.5g/kg, lard 0.5ml/kg) for 2 weeks, then rabbit abdominal aortic endothelium was damaged by using 4F Forgarty balloon catheter. After the arterial injury, high fat diet was maintained for 6 weeks and regular diet followed for 8 weeks. According to serum cholesterol level at 12 weeks, the rabbits were randomized into either a model group (n=6) or a simvastatin-treated (1mg/kg) group (n=6). A control group (n=6) was added simultaneously. After administration for 4 weeks, the rabbits were given ip RVV 0.12mg/kg and iv.histamine 0.016mg/kg, respectively.Twenty-four hours later, the rabbits were then sacrificed by bloodletting.
Measurement of thrombotic area:The artery was scissored from the aortic arch to the distal common iliac artery branches. Images of the arterial surface were taken and collected with digital camera (4 million pixels, Sony), the surface area covered by thrombus on atherosclerotic aorta was then determined and analyzed by a computer with a color image processing software of Sigma 4.0.
Morphologic observation:A 2 cm segment of the artery covered with atherosclerotic plaque was separated and impregnated with 10% formalin solution for paraffin slice and HE stain. Another 2cm fraction of the atherosclerotic artery was separated and fixed with 2.5% glutaral solution for electron microscope slice.
Measurement of cholesterol content:Fifty mg vessel of the atherosclerotic aorta was broken to pieces and homogenized in 1 ml extraction solution (chloroform: methanol=1∶1, v∶v ), the prepared samples were then filtrated with degreased filter paper, the filtrated solution was collected and added to 5 ml with extraction solution.
Cholesterol content per gram vessel tissue was measured by enzymology. Measurement of 6-keto-PGF1α and TXB2 in aorta:Fifty mg vessel of abdominal or thoracic aorta was incised into pieces respectively, then was put into a tissue grinder and triturated in 1 ml normal saline at 0℃. The homogenate was centrifuged at 1500×g for 10 min. Levels of 6-keto-PGF1α and TXB2 in aorta were determined by radioimmunoassay.
Hybridization in situ determination of MMP-2 mRNA expression 2cm portion of the atherosclerotic aorta was separated and fixed with 10% formalin solution for paraffin slice. MMP-2 mRNA expression was measured according to the operating manual of the hybridization in situ kit.
Statistical analysis:Data were expressed as x±s, and Students test was used for intergroup comparisons.
RESULTS
Changes of serum lipid and lipoprotein levels Serum TC, TG, LDL-C and HDL-C levels of pre-treatment in model and simvastatin-treated groups were observably increased as compared with control group(P<0.01). After treatment for 4 weeks, serum TC, TG, and LDL-C levels in simvastatin-treated group were lower than those in model group(P<0.01 or 0.05) (Table 1).
Effect on area of thrombosis:The results showed that the area of thrombus was very fewer in control group [mean surface area was (0.9±1.1)mm2] than that in model group [mean surface area was (78±53)mm2,P<0.01] or in simvastatin group [mean surface area was (17±12)mm2,P<0.01], but the area of simvastatin-treated group was significantly decreased as compared with model group (P<0.01, Fig.1 See Cover 4).
Effect on morphology of atherosclerotic aorta:Light microscopy showed that the structure of internal elastic membrane was integral, tunica media was full of smooth muscle cells in control group. Whereas in model group vessel showed inter-membrane incrassation, local ulcer in shoulder of the plaque, fibrin tissue hyperplasia and transparent denaturalization, and needle-like interspace that resulted from cholesterol crystal dissolved by solvent. Foam cells filled with lipid vacuole were rich in the plaque. Middle membrane under plaque was destructed in different degree, some turned thin due to atrophy. After treated with simvastatin for 4 weeks, light microscopy showed inter-membrane incrassation, fibrin tissue hyperplasia in plaque, fibrin cap incrassation. Most shoulders of plaques were more integrated, plaque ulcers were smaller and fewer, foam cells and needle-like interspace in plaque were also diminished (Fig.2 See Cover 4). Under electron microscopy, integrated endothelium, developed Golgi complex, rough endoplasmic reticulum, rich pinocytosis vesicle in plasma were visualized in control group. In model group, endothelium was destructed obviously, and endothelial cells could not be seen lining the intimal surface. Macrophages were richer and filled with lipid drops, some macrophages took on a fore-necrotic look including swell, membrane rupture, nucleus shrink and so on. Compared with model group, foam cells and lipid drops in simvastatin group were fewer and smaller relatively, macrophages were fewer, and showed mitochondrion and rough endoplasmic reticulum dilatation, lysosome increase, but cell structure was integrated relatively (Figure 3).
Figure 3 Electron micrographs of rabbit abdominal aorta. a：macrophage in model group, the arrow showed lipid drops and needle-like cholesterol crystal (×10000); b: simvastatin 1mg/kg treatment group, macrophage structure was integrated relatively (×10000)
Cholesterol content in abdominal aorta:Cholesterol content in model group was (24±8)mg/g wet tissue, and far higher than that in control group [(6.6±1.4)mg/g wet tissue,P<0.01] or in simvastatin group[(13±5)mg/g wet tissue,P<0.01)].
6-keto-PGF1α, TXB2 and their ratio in aorta:Abdominal aorta of atherosclerotic rabbits had been injured by balloon catheter, so its damnification was more serious than thoracic aorta correspondingly. The data indicated that 6-keto-PGF1αand TXB2 levels in aorta in model rabbits were increased, and their ratio was reduced as compared with control rabbits(P<0.01). Simvastatin might increase the ratio by increasing PGF1α in abdominal and thoracic aorta, and reducing TXB2 in thoracic aorta ( Table 2).
Expression of MMP-2 mRNA in abdominal aorta:Expression of MMP-2 mRNA in aorta was measured by hybridization in situ. Positive cell showed brown or brown-yellow granules. The results showed that cells in control group were no brown granules. In model group, there were many positive cells filled with brown granules, and distributed mainly under plaque and in shoulder of plaque. Simvastatin group expressed fewer positive cells, which distributed mainly under plaque and very few in the shoulder section. Positive cells of 5 eyeshot in every slide were counted in light microscope(×100). In model group the positive cells was 37±16,which was higher than that in simvastatin group (13±6)(P< 0.01).
Discussion
It is difficult to establish an animal model about atherosclerotic plaque rupture, though there are many ways to copy an animal model about atherosclerosis. The reason is mainly due to too many factors that affect plaque rupture. Now there are only two ways to establish animal model about plaque rupture[15, 25]. One is to find animals whose atherosclerotic plaque could be spontaneously ruptured, the other is to trigger plaque disruption in atherosclerotic animals. Spontaneous rupture is infrequent in atherosclerotic animals. In our study, we chose New Zealand white rabbits as experimental animal, the rabbits formed atherosclerosis after fed with high fat diet and undergone balloon-induced arterial injury. After the atherosclerosis developed, the plaque rupture was triggered by injection of RVV and histamine, the former is a proteolytic procoagulant and endothelial toxin, which could induce hypercoagulable state in the atherosclerotic rabbits, the latter is a vasopressor to rabbits, which could evoke plaque into rupture. Thus, plaque disruption and subsequent thrombosis produced in these atherosclerotic rabbits. In this study, thrombosis happened to most model animals, light microscopic examination of shoulder of plaque that adhered to thrombus showed obvious ulcer and inflammatory cell infiltration. The plaque was filled with many foam cells and lipid vacuoles. Under electron microscope, macrophages filled with lipid drops and cholesterol noodle-like crystal were abundant in plaque. Cell denaturalization was seen easily. The content of cholesterol in abdominal aorta increased observably. These features were in accordance with unstable plaque very well. Therefore we thought this animal model mimicked atherosclerotic plaque rupture preferably.
Simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, came into the market as a lipid-regulator for the first time, which could reduce blood cholesterol and LDL-C markedly[26-28]. Recently its non-lipid effects were identified. These effects included increasing synthesis of NO, decreasing synthesis of ET-1, inhibiting LDL-C oxidation, reducing inflammatory cells, C-reactive protein, inhibiting platelet adhesion / aggregation, reducing fibrinogen concentration and blood viscosity[29-31]. The present results showed that simvastatin could increase the plaque stability and decrease the thrombosis in atherosclerotic rabbits. The drug had a beneficial effect on atherosclerotic plaque stability, but its mechanism(s) is not known yet. In our study, we observed that simvastatin reduced cholesterol content of the plaque tissue, which could make lipid core turn smaller and increase plaque stability. Light microscope examination demonstrated that fibrin cap turned thicker, foam and inflammatory cells lessened obviously, shoulder section on plaque kept integrated correspondingly. The drug also inhibited the expression of MMP-2 mRNA in atherosclerotic tissue. Being short of MMPs, fibrin cap of plaque is analyzed reductively and the plaque turns solid. In addition, some inflammatory factors may affect coagulation system. For instance, PGI2 vasodilates blood vessel and inhibits thrombosis, but TXA2 contracts artery and promotes thrombosis, its ratio is vital to thrombosis forming. In our study, we have observed that simvastatin promoted 6-keto-PGF1α/TXB2 ratio in abdominal and thoracic aorta of atherosclerotic rabbits, which was one of the possible mechanisms of reducing thrombosis.
In summary, the mechanisms of increasing stability of atherosclerotic plaque and inhibiting thrombosis by simvastatin may be associated with lipid-regulation and many non-lipid effects, which requires to be investigated further.
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Supported by Soochow University Medical Development Foundation, No.010721
1.Department of Pharmacology, Medical School of Soochow University,Suzhou,Jiangsu Province 215123,China
2.Taizhou Hospital ,Linhai,Zhejiang Province 317000，China
*Corresponding to Professor Xie Mei-Lin, Tel: +86-512-6588-0320,E-mail address: xiemeilin@suda.edu.cn
(Editor Hu Dan)ARTICLES(CAI Bao-xiang, XIE Mei-li)
【Key words】 simvastatin; plaque stability; thrombosis; rabbits
INTRODUCTION
Cardio-cerebral vascular disease is the leading cause of mortality in many countries and carries an important socioeconomic burden. Atherosclerosis is the main pathologic basis of cardio-cerebral vascular disease. With the development of atherosclerosis, plaque rupture and thrombosis will occur, that is critical to the onset of stroke, acute coronary ischemic syndromes[1-3] and so on. The vulnerable plaque is generally composed of an atrophic fibrous cap, a lipid-rich necrotic core, and accumulation of inflammatory cells[4-7]. Erosion of the fibrous cap, usually correlating to matrix metalloproteinases (MMPs) [8,9] such as MMP-2, often leads to rupture of the plaque, with subsequent occlusive thrombus formation, which relates to the imbalance between PGI2 and TXA2[10-13].PGI2 generated by vascular endotheliocytes, is a potent platelet inhibitor and vasodilator, and TXA2, released from activated platelets, is a potent vasoconstrictor and platelet-aggregating agent. Recent research has identified that the stability of plaque is more significant than its absolute size[14-16], so it is crucial to increase plaque stability to prevent acute cardiovascular and cerebrovascular diseases.
Statins such as simvastatin, a kind of lipidemic regulators, are widely used for the treatment of hypercholesterolaemia. In recent years, however, there has been an increasing body of evidence that their effects on lipid profile cannot fully account for their cardiovascular protective actions[17-20]: their beneficial effects are too rapid to be easily explained by their relatively slow effects on atherogenesis and too large to be accounted for by their relatively small effects on plaque regression.Therefore,it is now thought that their beneficial effects on cardio-cerebral vascular system might result from improving vascular endothelial function, inhibiting platelet aggregation, and reducing inflammatory cell accumulation[21-24]. These findings of clinical observation and cell culture prompted us to investigate whether statins increased plaque stability in animal model of atherosclerotic plaque rupture.
The present studies were designed to address the following three questions. First, we wished to determine whether simvastatin had any effect on reducing thrombotic area in rabbit atherosclerotic plaque rupture model. Secondly, we wished to evaluate changes in the morphological features related to plaque instability after treatment of atherosclerotic rabbits with simvastatin. Finally, we wished to determine whether simvastatin had any effect on TXA2, PGI2 and MMP-2 in aortic vessel tissue. The data demonstrated that simvastatin not only reduced lipid levels, but also inhibited plaque repture and subsequent thrombosis, its mechanisms might include decreasing inflammatory cell and lipid infiltration, MMP-2 mRNA expression and TXA2 formation, increasing PGI2 content and PGI2/TXA2 ratio in the artery covered with atherosclerotic plaque.
MATERIALS AND METHODS
Animals:Male New Zealand white rabbits, weighing (2.0±0.2)kg were provided by Experimental Animal Center of Soochow University (Certificate No: YCYK 2002-0008).
Drugs and reagents:Simvastatin was provided by Hangzhou Merck Sharp & Dohme Pharmaceutical Co. Ltd, China. Russells viper venom (RVV) and histamine were products of Sigma Chemical Co，USA. Cholesterol was a product of Nanjing Xinbai Pharmaceutical Co., China. TC, TG, high density lipoprotein-cholesterol (HDL-C) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. MMP-2 mRNA in situ hybridization detection kit was supplied by Wuhan Boshide Bioengineering Co. Ltd, China. 6-keto-PGF1α and TXB2 radioimmunity assay kits were supplied by Radioimmunity Institute of Soochow University.
Animal model preparation:Experimental rabbits were fed with a high fat diet (cholesterol 0.5g/kg, lard 0.5ml/kg) for 2 weeks, then rabbit abdominal aortic endothelium was damaged by using 4F Forgarty balloon catheter. After the arterial injury, high fat diet was maintained for 6 weeks and regular diet followed for 8 weeks. According to serum cholesterol level at 12 weeks, the rabbits were randomized into either a model group (n=6) or a simvastatin-treated (1mg/kg) group (n=6). A control group (n=6) was added simultaneously. After administration for 4 weeks, the rabbits were given ip RVV 0.12mg/kg and iv.histamine 0.016mg/kg, respectively.Twenty-four hours later, the rabbits were then sacrificed by bloodletting.
Measurement of thrombotic area:The artery was scissored from the aortic arch to the distal common iliac artery branches. Images of the arterial surface were taken and collected with digital camera (4 million pixels, Sony), the surface area covered by thrombus on atherosclerotic aorta was then determined and analyzed by a computer with a color image processing software of Sigma 4.0.
Morphologic observation:A 2 cm segment of the artery covered with atherosclerotic plaque was separated and impregnated with 10% formalin solution for paraffin slice and HE stain. Another 2cm fraction of the atherosclerotic artery was separated and fixed with 2.5% glutaral solution for electron microscope slice.
Measurement of cholesterol content:Fifty mg vessel of the atherosclerotic aorta was broken to pieces and homogenized in 1 ml extraction solution (chloroform: methanol=1∶1, v∶v ), the prepared samples were then filtrated with degreased filter paper, the filtrated solution was collected and added to 5 ml with extraction solution.
Cholesterol content per gram vessel tissue was measured by enzymology. Measurement of 6-keto-PGF1α and TXB2 in aorta:Fifty mg vessel of abdominal or thoracic aorta was incised into pieces respectively, then was put into a tissue grinder and triturated in 1 ml normal saline at 0℃. The homogenate was centrifuged at 1500×g for 10 min. Levels of 6-keto-PGF1α and TXB2 in aorta were determined by radioimmunoassay.
Hybridization in situ determination of MMP-2 mRNA expression 2cm portion of the atherosclerotic aorta was separated and fixed with 10% formalin solution for paraffin slice. MMP-2 mRNA expression was measured according to the operating manual of the hybridization in situ kit.
Statistical analysis:Data were expressed as x±s, and Students test was used for intergroup comparisons.
RESULTS
Changes of serum lipid and lipoprotein levels Serum TC, TG, LDL-C and HDL-C levels of pre-treatment in model and simvastatin-treated groups were observably increased as compared with control group(P<0.01). After treatment for 4 weeks, serum TC, TG, and LDL-C levels in simvastatin-treated group were lower than those in model group(P<0.01 or 0.05) (Table 1).
Effect on area of thrombosis:The results showed that the area of thrombus was very fewer in control group [mean surface area was (0.9±1.1)mm2] than that in model group [mean surface area was (78±53)mm2,P<0.01] or in simvastatin group [mean surface area was (17±12)mm2,P<0.01], but the area of simvastatin-treated group was significantly decreased as compared with model group (P<0.01, Fig.1 See Cover 4).
Effect on morphology of atherosclerotic aorta:Light microscopy showed that the structure of internal elastic membrane was integral, tunica media was full of smooth muscle cells in control group. Whereas in model group vessel showed inter-membrane incrassation, local ulcer in shoulder of the plaque, fibrin tissue hyperplasia and transparent denaturalization, and needle-like interspace that resulted from cholesterol crystal dissolved by solvent. Foam cells filled with lipid vacuole were rich in the plaque. Middle membrane under plaque was destructed in different degree, some turned thin due to atrophy. After treated with simvastatin for 4 weeks, light microscopy showed inter-membrane incrassation, fibrin tissue hyperplasia in plaque, fibrin cap incrassation. Most shoulders of plaques were more integrated, plaque ulcers were smaller and fewer, foam cells and needle-like interspace in plaque were also diminished (Fig.2 See Cover 4). Under electron microscopy, integrated endothelium, developed Golgi complex, rough endoplasmic reticulum, rich pinocytosis vesicle in plasma were visualized in control group. In model group, endothelium was destructed obviously, and endothelial cells could not be seen lining the intimal surface. Macrophages were richer and filled with lipid drops, some macrophages took on a fore-necrotic look including swell, membrane rupture, nucleus shrink and so on. Compared with model group, foam cells and lipid drops in simvastatin group were fewer and smaller relatively, macrophages were fewer, and showed mitochondrion and rough endoplasmic reticulum dilatation, lysosome increase, but cell structure was integrated relatively (Figure 3).
Figure 3 Electron micrographs of rabbit abdominal aorta. a：macrophage in model group, the arrow showed lipid drops and needle-like cholesterol crystal (×10000); b: simvastatin 1mg/kg treatment group, macrophage structure was integrated relatively (×10000)
Cholesterol content in abdominal aorta:Cholesterol content in model group was (24±8)mg/g wet tissue, and far higher than that in control group [(6.6±1.4)mg/g wet tissue,P<0.01] or in simvastatin group[(13±5)mg/g wet tissue,P<0.01)].
6-keto-PGF1α, TXB2 and their ratio in aorta:Abdominal aorta of atherosclerotic rabbits had been injured by balloon catheter, so its damnification was more serious than thoracic aorta correspondingly. The data indicated that 6-keto-PGF1αand TXB2 levels in aorta in model rabbits were increased, and their ratio was reduced as compared with control rabbits(P<0.01). Simvastatin might increase the ratio by increasing PGF1α in abdominal and thoracic aorta, and reducing TXB2 in thoracic aorta ( Table 2).
Expression of MMP-2 mRNA in abdominal aorta:Expression of MMP-2 mRNA in aorta was measured by hybridization in situ. Positive cell showed brown or brown-yellow granules. The results showed that cells in control group were no brown granules. In model group, there were many positive cells filled with brown granules, and distributed mainly under plaque and in shoulder of plaque. Simvastatin group expressed fewer positive cells, which distributed mainly under plaque and very few in the shoulder section. Positive cells of 5 eyeshot in every slide were counted in light microscope(×100). In model group the positive cells was 37±16,which was higher than that in simvastatin group (13±6)(P< 0.01).
Discussion
It is difficult to establish an animal model about atherosclerotic plaque rupture, though there are many ways to copy an animal model about atherosclerosis. The reason is mainly due to too many factors that affect plaque rupture. Now there are only two ways to establish animal model about plaque rupture[15, 25]. One is to find animals whose atherosclerotic plaque could be spontaneously ruptured, the other is to trigger plaque disruption in atherosclerotic animals. Spontaneous rupture is infrequent in atherosclerotic animals. In our study, we chose New Zealand white rabbits as experimental animal, the rabbits formed atherosclerosis after fed with high fat diet and undergone balloon-induced arterial injury. After the atherosclerosis developed, the plaque rupture was triggered by injection of RVV and histamine, the former is a proteolytic procoagulant and endothelial toxin, which could induce hypercoagulable state in the atherosclerotic rabbits, the latter is a vasopressor to rabbits, which could evoke plaque into rupture. Thus, plaque disruption and subsequent thrombosis produced in these atherosclerotic rabbits. In this study, thrombosis happened to most model animals, light microscopic examination of shoulder of plaque that adhered to thrombus showed obvious ulcer and inflammatory cell infiltration. The plaque was filled with many foam cells and lipid vacuoles. Under electron microscope, macrophages filled with lipid drops and cholesterol noodle-like crystal were abundant in plaque. Cell denaturalization was seen easily. The content of cholesterol in abdominal aorta increased observably. These features were in accordance with unstable plaque very well. Therefore we thought this animal model mimicked atherosclerotic plaque rupture preferably.
Simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, came into the market as a lipid-regulator for the first time, which could reduce blood cholesterol and LDL-C markedly[26-28]. Recently its non-lipid effects were identified. These effects included increasing synthesis of NO, decreasing synthesis of ET-1, inhibiting LDL-C oxidation, reducing inflammatory cells, C-reactive protein, inhibiting platelet adhesion / aggregation, reducing fibrinogen concentration and blood viscosity[29-31]. The present results showed that simvastatin could increase the plaque stability and decrease the thrombosis in atherosclerotic rabbits. The drug had a beneficial effect on atherosclerotic plaque stability, but its mechanism(s) is not known yet. In our study, we observed that simvastatin reduced cholesterol content of the plaque tissue, which could make lipid core turn smaller and increase plaque stability. Light microscope examination demonstrated that fibrin cap turned thicker, foam and inflammatory cells lessened obviously, shoulder section on plaque kept integrated correspondingly. The drug also inhibited the expression of MMP-2 mRNA in atherosclerotic tissue. Being short of MMPs, fibrin cap of plaque is analyzed reductively and the plaque turns solid. In addition, some inflammatory factors may affect coagulation system. For instance, PGI2 vasodilates blood vessel and inhibits thrombosis, but TXA2 contracts artery and promotes thrombosis, its ratio is vital to thrombosis forming. In our study, we have observed that simvastatin promoted 6-keto-PGF1α/TXB2 ratio in abdominal and thoracic aorta of atherosclerotic rabbits, which was one of the possible mechanisms of reducing thrombosis.
In summary, the mechanisms of increasing stability of atherosclerotic plaque and inhibiting thrombosis by simvastatin may be associated with lipid-regulation and many non-lipid effects, which requires to be investigated further.
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Supported by Soochow University Medical Development Foundation, No.010721
1.Department of Pharmacology, Medical School of Soochow University,Suzhou,Jiangsu Province 215123,China
2.Taizhou Hospital ,Linhai,Zhejiang Province 317000，China
*Corresponding to Professor Xie Mei-Lin, Tel: +86-512-6588-0320,E-mail address: xiemeilin@suda.edu.cn
(Editor Hu Dan)ARTICLES(CAI Bao-xiang, XIE Mei-li)